During vertebrate spermatogenesis, chromatin is dramatically reorganized in developing spermatids via histone replacement with protamines to achieve an even more compacted state for efficient genetic delivery and DNA protection. Dense packaging of DNA in sperm nuclei is considered important to protect the DNA against damage by mutagens or oxidative species. Protamines are small, arginine-rich, nuclear proteins that condense the spermatid genome into a genetically inactive state. Salmon protamine is a 32 amino acid peptide with 21 arginines and 11 neutral amino acids (PR4S3RPVR5PRVSR6G2R4). Unlike histone compacted DNA, the physical properties of reconstituted of protamine - DNA assemblies closely resemble those of DNA condensed by multivalent cations that are much smaller, have much less charge, and have been better characterized such as divalent manganese, cobalt hexammine, spermidine, spermine, and several arginine oligopeptides. DNA is packaged by protamine to densities within the range seen for DNA condensed with the smaller multivalent ions. More importantly, the forces measured for both reconstituted protamine DNA arrays and native salmon nuclei by the osmotic stress technique coupled with x-ray diffraction show very similar characteristics to DNA precipitated by multivalent ions. We hypothesize that male infertility from protamine deficiency and errors in modification is due to looser packing of DNA in sperm nuclei than is optimal and hence greater accessibility to mutagens and oxidizing species. Upon condensation with the smaller multivalent ions or protamine, the resulting compacted structures have well-defined equilibrium separations of the DNA double helices of 7 - 15 , depending on the identity of the condensing ion. The finite separation of helices indicates a delicate balancing of a short ranged repulsive force with a longer ranged attraction. The physical origins of the forces acting to compact multivalent ion - DNA assemblies are still debated. We have argued for water-structuring or hydration forces on the basis of our previous extensive measurements of forces between charged and uncharged molecules. Most theories of DNA assembly include some form of correlation of positive charges on one helix with negative phosphate charges on another in order to account for attraction. Using combined osmotic stress and single molecule tweezing experiments, we have previously characterized the distance dependence of the two component forces. The attractive force varies with DNA-DNA spacing as a 4 - 5 decay length exponential;whereas the repulsive force is a 2 - 2.5 decay length exponential. The factor of two in decay lengths suggests that the attractive force results from a direct interaction of charged groups on apposing helices;whereas the repulsive force seems to be an image-charge or its hydration equivalent force. Using this constraint, we can effectively separate the attractive and repulsive contributions to the force - distance curves. The amplitudes of the two forces were extracted from the force curves for a set of homologous arginine peptides, R1 through R6 and poly-arginine. The magnitude of the attractive force depends on the arginine peptide charge. The repulsive force amplitude, in contrast, is nearly independent of arginine peptide length. The equilibrium spacing between DNA helices in reconstituted +21 charged salmon protamine DNA arrays was only equivalent to +5 charged R5. This is surprising since it is generally considered that cation charge is the dominating determinant of DNA compaction. The separation of the forces for salmon protamine-DNA assemblies indicated that the attractive force amplitude with protamine was very close to that expected for 21 arginines, but that the repulsive force had a larger amplitude than expected from the homo-arginine peptide series. This additional repulsion results in the observed lower packaging efficiency of +21 charged protamine compared to +6 hexa-arginine. Salmon protamine is long enough that the extra repulsion could be due to defects in its binding along the DNA helix. In order to determine if this extra repulsion is a general property of including neutral amino acids in arginine based peptides or if it is something peculiar to protamine, we investigated the effect on forces of inserting neutral amino acids into model hexa-arginine (R6) peptides. Neutral amino acids inserted into R6 significantly increase the amplitude of short ranged repulsive force. We observe a smaller decrease in the amplitude of the attraction. The effect on force amplitudes of incorporating increasing numbers of alanines into the middle of R6 is additive at least through four. There is relatively little difference in inserting alanine, serine, proline, or isoleucine. We find that the increase in the short ranged repulsive force for salmon protamine condensed DNA is in reasonablequantitative agreement with its neutral amino acid content. We also investigated the effect of incorporating a single negative charge, glutamate or phosphorylated serine, into R6. The net attraction between DNA helices is substantially weakened, much more than simply reducing the net charge by one. Indeed, the equilibrium spacing is significantly greater that seen with R3. The short-ranged repulsion is dramatically increased. The longer ranged attraction is moderately decreased, but is consistent with a net +5 charge. There are several differences between mammalian and piscine protamines. Most but not all mammals have two protamines, P1 and P2, both of which are longer than piscine protamines. The average fraction arginine of mammalian protamines, 50-60%, is significantly smaller than for fish, 65-70%. On the basis of our results, the increase in neutral amino acid content would be expected to decrease the net attraction, significantly increasing the equilibrium spacing between helices in mammalian sperm. A looser packaging of DNA due to an increased fraction of neutral amino acids would increase accessibility of mutagens and reactive oxidizing species to the DNA causing increased damage. This might explain another difference between mammalian and piscine protamine-DNA packaging. Extensive disulfide bridges between protamines are present in mammalian sperm. Most piscine protamines, on the other hand, do not have cysteines. The disulfide bridges in mammals may be required for tight DNA packaging, overcoming the increased fraction of neutral amino acids. Not surprisingly, the incorporation of a negatively charged amino acid into R6 has a large effect on forces. This likely has biological implications for spermatogenesis. The replacement of histones by protamines occurs in several steps. First acetylated histones are replaced by transition proteins. Serine phosphorylated protamines than replace the transition proteins. It is only after removing the phosphate groups that DNA is tightly packed. Incomplete dephosphorylation has been suggested a cause of increased DNA damage and male infertility. The results here show that phosphorylation has a much larger effect on weakening the net attraction between helices than simply reducing the protamine charge would suggest. We have recently begun examination of mammalian sperm nuclei and are still optimizing techniques. Our preliminary data indicates that with intact disulfide bridges stallion sperm DNA is packaged as tightly as in salmon sperm. The spacing between helices in stallion sperm greatly increases when the disulfide bridges are reduced in agreement with our expectations. We expect to find a correlation between DNA packing densities and infertility.